New Indazol-Pyrimidine-Based Derivatives as Selective Anticancer Agents: Design, Synthesis, and In Silico Studies

In this research study, the authors successfully synthesized potent new anticancer agents derived from indazol-pyrimidine. All the prepared compounds were tested for in vitro cell line inhibitory activity against three different cancerous cell lines. Results demonstrated that five of the novel compounds—4f, 4i, 4a, 4g, and 4d—possessed significant cytotoxic inhibitory activity against the MCF-7 cell line, with IC50 values of 1.629, 1.841, 2.958, 4.680, and 4.798 μM, respectively, compared to the reference drug with an IC50 value of 8.029 μM, thus demonstrating promising suppression power. Compounds 4i, 4g, 4e, 4d, and 4a showed effective cytotoxic activity stronger than the standard against Caco2 cells. Moreover, compounds 4a and 4i exhibited potent antiproliferative activity against the A549 cell line that was stronger than the reference drug. The most active products, 4f and 4i, werr e further examined for their mechanism of action. It turns out that they were capable of activating caspase-3/7 and, therefore, inducing apoptosis. However, produced a higher safety profile than the reference drug, towards the normal cells (MCF10a). Furthermore, the dynamic nature, binding interaction, and protein–ligand stability were explored through a Molecular Dynamics (MD) simulation study. Various analysis parameters (RMSD, RMSF, RoG, and SASA) from the MD simulation trajectory have suggested the stability of the compounds during the 20 ns MD simulation study. In silico ADMET results revealed that the synthesized compounds had low toxicity, good solubility, and an absorption profile since they met Lipinski’s rule of five and Veber’s rule. The present research highlights the potential of derivatives with indazole scaffolds bearing pyrimidine as a lead compound for designing anticancer agents.


Introduction
Cancer is a polygenic disease and one of the major aggressive health problems facing humans [1]. A report published by GLOBOCAN 2020 estimated that 19.3 million cancer cases and almost 10.0 million cancer deaths occurred in 2020 [2]. High mortality rates were reported in lung and colorectal cancer globally, in addition to breast cancer [3]. Treatment of cancer causes a state of extreme physical or mental fatigue until now due to its strong toxicity and the lack of efficiency of commercial anticancer drugs. In recent years, effective targeting has led to the development of more efficient and less toxic anticancer agents [4]. Pyrimidine derivatives are one of the most effective bioactive agents that exhibit a wide range of medicinal applications because they are essential parts of the nucleic acids that make up DNA and RNA [5]. Pyrimidine derivatives are heterocyclic scaffolds that represent one of the most bioactive pharmacophores implemented as anticancer agents [6][7][8][9] by using different mechanisms. For example, pyrimidines act as selective dual inhibitors of make up DNA and RNA [5]. Pyrimidine derivatives are heterocyclic scaffolds that represent one of the most bioactive pharmacophores implemented as anticancer agents [6][7][8][9] by using different mechanisms. For example, pyrimidines act as selective dual inhibitors of c-Met and VEGFR-2 [10], dual ERα/VEGFR-2 ligands with anti-breast cancer activity [11], and selective inhibitors against triple-negative breast cancer cell line MDA-MB-468 [12], and they have antiproliferative activity and EGFR and ARO inhibitory activity [13]. Furthermore, some pyrimidines act by inhibiting different proteins and enzymes that play key roles in the cell cycle and division [14]. Also, they act as antituberculosis agents and antimicrobials [15,16]. On the other hand, indazole derivatives are one of the most outstanding scaffolds and have a wide range of properties, including anti-inflammatory, anti-HIV, antiplatelet, and serotonin 5-HT3 receptor antagonist characteristics [17][18][19]. Peerreviewed literature has reported that indazoles possess antibacterial, anti-inflammatory, antitubercular, and antidepressant traits [20][21][22]. Indazole derivatives exhibit potential anticancer activity, which makes them useful scaffolds for the development of new anticancer agents [23]. Indazole-N-phenylpyrimidin-2-amine derivatives I were synthesized and demonstrated bioactive products by Pawel M. et al. (Figure 1) [24]. CYC116 (Figure 1, II) is a novel anticancer molecule targeting both the cell cycle and angiogenesis, with antitumor activity in both solid tumors and hematological cancers [25]. Elsayed et al. [26] synthesized new indazole-pyrimidines ( Figure 1, compound III) with potent antigenic effects against VEGFR kinase. Moreover, a sulfonamide moiety incorporated into different heterocyclic ring systems has been reported as one of the most specific scaffolds to inhibit the growth of different types of human cancer cells [27,28]. Thus, both pyrimidine and indazole derivatives represent privileged scaffolds in medicinal chemistry. Our strategy is to incorporate these two heterocyclic moieties in one molecule through an amino and/or sulfonamide linkage to synthesize new indazol-pyrimidine derivatives and evaluate their antiproliferative activity using an MTT assay against MCF-7, A549, and Caco2 cell lines. The most promising candidates were examined for their mechanism of action, their effect on the cell cycle, and their apoptosis stimulation potential on cancer cells, followed by their effect on normal cells. We are hoping to construct new bioactive heterocyclic hybrids that may enhance or increase the biological activities of the new products and may be useful scaffolds for developing new effective anticancer agents with high safety profiles. Finally, in silico studies, including molecular docking and drug-likeness studies, were performed.  of 5-aminoindazole 2 with 2,4-dichloropyrimidine 1a or 5-flouro-2,4 dichloropyrimidine 1b. The chlorine atom at position 4 of compound 1a or 1b was reacted with the amino group at position 5 of 5-aminoindazol 2 regio-selectively. Compounds 3a and b were first synthesized by refluxing a mixture of 5-substituted-2,4-dichloropyrimidine 1a or 1b with 5-aminoindazole 2 using equimolar concentrations in ethanol and a few drops of HCl. However, impure compounds and poor yields were obtained using this method. Thus, another method was used by replacing HCl with triethylamine, yielding pure products with good yields [26]. The presence of the electronegative fluorine moiety of compound 1b reduced the reaction time to around three hours, while the presence of hydrogen at the same position resulted in a longer reaction time (~12 h). Another nucleophilic substitution reaction took place to synthesize compounds 4a-i, by the substitution of the Cl atom at position 2 of the pyrimidine ring in compounds 3a and b with the amino group of the aniline derivatives, by refluxing the appropriate aniline with compound 3a or 3b in butanol and a few drops of HCl to yield the target compounds 4a-i; Scheme 1. All the used anilines were commercially available except the morpholino aniline, which was synthesized by reduction of the nitro group of compound 6 to afford the morpholino-aniline derivative 7, according to the reported method [29] as presented in Scheme 2. In general, target compounds having an F atom at position 5 of the pyrimidine ring were obtained faster and with a better yield, which might be due to its inductive electron-withdrawing effect. Meanwhile, poorer yields were obtained from compounds having hydrogen atoms at the same position. In addition, further mechanistic studies were performed to investigate the mode of cell death and cell cycle changes.
The chlorine atom at position 4 of compound 1a or 1b was reacted with the amino group at position 5 of 5-aminoindazol 2 regio-selectively. Compounds 3a and b were first synthesized by refluxing a mixture of 5-substituted-2,4-dichloropyrimidine 1a or 1b with 5aminoindazole 2 using equimolar concentrations in ethanol and a few drops of HCl. However, impure compounds and poor yields were obtained using this method. Thus, another method was used by replacing HCl with triethylamine, yielding pure products with good yields [26]. The presence of the electronegative fluorine moiety of compound 1b reduced the reaction time to around three hours, while the presence of hydrogen at the same position resulted in a longer reaction time (~12 h). Another nucleophilic substitution reaction took place to synthesize compounds 4a-i, by the substitution of the Cl atom at position 2 of the pyrimidine ring in compounds 3a and b with the amino group of the aniline derivatives, by refluxing the appropriate aniline with compound 3a or 3b in butanol and a few drops of HCl to yield the target compounds 4a-i; Scheme 1. All the used anilines were commercially available except the morpholino aniline, which was synthesized by reduction of the nitro group of compound 6 to afford the morpholino-aniline derivative 7, according to the reported method [29] as presented in Scheme 2. In general, target compounds having an F atom at position 5 of the pyrimidine ring were obtained faster and with a better yield, which might be due to its inductive electron-withdrawing effect. Meanwhile, poorer yields were obtained from compounds having hydrogen atoms at the same position. In addition, further mechanistic studies were performed to investigate the mode of cell death and cell cycle changes.
The chlorine atom at position 4 of compound 1a or 1b was reacted with the amino group at position 5 of 5-aminoindazol 2 regio-selectively. Compounds 3a and b were first synthesized by refluxing a mixture of 5-substituted-2,4-dichloropyrimidine 1a or 1b with 5aminoindazole 2 using equimolar concentrations in ethanol and a few drops of HCl. However, impure compounds and poor yields were obtained using this method. Thus, another method was used by replacing HCl with triethylamine, yielding pure products with good yields [26]. The presence of the electronegative fluorine moiety of compound 1b reduced the reaction time to around three hours, while the presence of hydrogen at the same position resulted in a longer reaction time (~12 h). Another nucleophilic substitution reaction took place to synthesize compounds 4a-i, by the substitution of the Cl atom at position 2 of the pyrimidine ring in compounds 3a and b with the amino group of the aniline derivatives, by refluxing the appropriate aniline with compound 3a or 3b in butanol and a few drops of HCl to yield the target compounds 4a-i; Scheme 1. All the used anilines were commercially available except the morpholino aniline, which was synthesized by reduction of the nitro group of compound 6 to afford the morpholino-aniline derivative 7, according to the reported method [29] as presented in Scheme 2. In general, target compounds having an F atom at position 5 of the pyrimidine ring were obtained faster and with a better yield, which might be due to its inductive electron-withdrawing effect. Meanwhile, poorer yields were obtained from compounds having hydrogen atoms at the same position. In addition, further mechanistic studies were performed to investigate the mode of cell death and cell cycle changes.

MTT Cytotoxicity Assay
All of the synthesized compounds were screened for their cytotoxic effect against three different human cancer cells, namely breast cancer cells (MCF-7), lung cancer cells (A549), and colorectal adenocarcinoma cells (Caco2), using the MTT cytotoxic assay [30,31]. The results are summarized in Table 1, demonstrating that five compounds-4f, 4i, 4a, 4d, and 4g-had potent cytotoxic activity against MCF-7 cells and were more active than the reference drug, Staurosporine. Compounds 4f, 4i, and 4a demonstrated the strongest cytotoxic effect, with IC 50 values of 1.629, 1.841, and 2.958 µM, respectively, compared to the reference (IC 50 Table 2). The results showed that their IC 50 values (23.67 and 29.5 µM, respectively) against normal cells demonstrated marked safety profiles toward human normal cells, more than the standard drug (IC 50 = 34.8 µM) by 10 and 5 units for 4f and 4i, respectively. Therefore, these compounds were more selective toward cancerous cells, with selectivity index values of IS 14.5 and 16.03, respectively, relative to the standard drug with IS 4.34. An encouraging strategy for cancer therapy is targeting the cell cycle, according to the reported method [32]. In this study, compounds 4i and 4f were tested-using a DNA flow cytometry assay-for their effects on the cell cycle of MCF-7 cells (Figure 2). Results showed that after treating MCF-7 cells with compound 4i or 4f for 24 h, a slight increase in the percentage of cells in the G0-G1 phase for both compounds was observed-around 7% compared with the control (53.89%). However, compound 4i exhibited a slight increase (3%) in the percentage of the cell population in the S phase. Simultaneously, there was a significant reduction in the percentage of the cell population in the G2/M phase-32% and 46% for cells treated with compound 4f or 4i, respectively, compared with controls. The decline in the proportion of cells in the G2/M phase as well as the increase in the cell population in the G0-G1 and S phases indicate a reduction in cell cycle progression in MCF-7 cells. An encouraging strategy for cancer therapy is targeting the cell cycle, according to the reported method [32]. In this study, compounds 4i and 4f were tested-using a DNA flow cytometry assay-for their effects on the cell cycle of MCF-7 cells ( Figure 2). Results showed that after treating MCF-7 cells with compound 4i or 4f for 24 h, a slight increase in the percentage of cells in the G0-G1 phase for both compounds was observed-around 7% compared with the control (53.89%). However, compound 4i exhibited a slight increase (3%) in the percentage of the cell population in the S phase. Simultaneously, there was a significant reduction in the percentage of the cell population in the G2/M phase-32% and 46% for cells treated with compound 4f or 4i, respectively, compared with controls. The decline in the proportion of cells in the G2/M phase as well as the increase in the cell population in the G0-G1 and S phases indicate a reduction in cell cycle progression in MCF-7 cells.

Apoptosis Induction and Caspase-3/7 Activation
To assess the apoptotic potential, MCF-7 cells were treated with compound 4i or 4f for 24 h and then assayed for Annexin-V/PI (propidium iodide) binding according to the reported protocol [31]. Results are displayed in Figure 3, which show that, these compounds increased the percentage of Annexin V/PI-stained cells to about 47% in both early and late phases of apoptosis, compared with 1.76% of the untreated cells. At the early phase of apoptosis, the average increment for the treated groups was around 27%, while it was around 14% in late-stage apoptosis.

Apoptosis Induction and Caspase-3/7 Activation
To assess the apoptotic potential, MCF-7 cells were treated with compound 4i or 4f for 24 h and then assayed for Annexin-V/PI (propidium iodide) binding according to the reported protocol [31]. Results are displayed in Figure 3, which show that, these compounds increased the percentage of Annexin V/PI-stained cells to about 47% in both early and late phases of apoptosis, compared with 1.76% of the untreated cells. At the early phase of apoptosis, the average increment for the treated groups was around 27%, while it was around 14% in late-stage apoptosis. An encouraging strategy for cancer therapy is targeting the cell cycle, according to the reported method [32]. In this study, compounds 4i and 4f were tested-using a DNA flow cytometry assay-for their effects on the cell cycle of MCF-7 cells ( Figure 2). Results showed that after treating MCF-7 cells with compound 4i or 4f for 24 h, a slight increase in the percentage of cells in the G0-G1 phase for both compounds was observed-around 7% compared with the control (53.89%). However, compound 4i exhibited a slight increase (3%) in the percentage of the cell population in the S phase. Simultaneously, there was a significant reduction in the percentage of the cell population in the G2/M phase-32% and 46% for cells treated with compound 4f or 4i, respectively, compared with controls. The decline in the proportion of cells in the G2/M phase as well as the increase in the cell population in the G0-G1 and S phases indicate a reduction in cell cycle progression in MCF-7 cells.

Apoptosis Induction and Caspase-3/7 Activation
To assess the apoptotic potential, MCF-7 cells were treated with compound 4i or 4f for 24 h and then assayed for Annexin-V/PI (propidium iodide) binding according to the reported protocol [31]. Results are displayed in Figure 3, which show that, these compounds increased the percentage of Annexin V/PI-stained cells to about 47% in both early and late phases of apoptosis, compared with 1.76% of the untreated cells. At the early phase of apoptosis, the average increment for the treated groups was around 27%, while it was around 14% in late-stage apoptosis.  Caspases are essential factors in apoptotic cell death since they play a key role in maintaining homeostasis, and their activation induces apoptosis. Caspase-3/7 activity results are displayed in Figure 4. When compared with untreated controls, the levels of active caspase-3/7 expression in MCF-7 cells increased from 0.43% to 19% and 26.5% after being treated with compound 4f or 4i, respectively. This demonstrates the potential apoptotic effect of compounds 4f and 4i in MCF-7 cells. Caspases are essential factors in apoptotic cell death since they play a key role in maintaining homeostasis, and their activation induces apoptosis. Caspase-3/7 activity results are displayed in Figure 4. When compared with untreated controls, the levels of active caspase-3/7 expression in MCF-7 cells increased from 0.43% to 19% and 26.5% after being treated with compound 4f or 4i, respectively. This demonstrates the potential apoptotic effect of compounds 4f and 4i in MCF-7 cells.

Molecular Dynamic and System Stability
A molecular dynamic simulation was carried out to predict the performance of the extracted compounds upon binding to the active site of protein as well as its interaction and stability through simulation [33,34]. The validation of system stability is essential to trace disrupted motions and avoid artifacts that may develop during the simulation. This study assessed Root-Mean-Square Deviation (RMSD) to measure system stability during the 20 ns simulations. The recorded average RMSD values for all frames of systems-apoprotein, 4f-complex, and 4i-complex systems-were 2.92 ± 0.56 Å, 2.04 ± 0.41 Å, and 2.49 ± 0.34 Å, respectively ( Figure 5A). These results revealed that the 4f-bound-to-protein complex system acquired a relatively more stable conformation than the other studied systems. During MD simulation, assessing protein structural flexibility upon ligand binding is critical for examining residue behavior and its connection with the ligand [35]. Protein residue fluctuations were evaluated using the Root-Mean-Square Fluctuation (RMSF) algorithm to evaluate the effect of inhibitor binding toward the respective targets over 20 ns simulations. The computed average RMSF values were 6.24 Å, 0.99 Å, and 4.70 Å for apo-protein, 4f-complex, and 4i-complex systems, respectively. Overall residue fluctuations of individual systems are represented in Figure 5B. These values revealed that the 4f-bound-to-protein complex system has a lower residue fluctuation than the other systems. ROG was determined to evaluate overall system compactness as well as stability upon ligand binding during MD simulation [36,37]. The average Rg values for apo-protein, 4f-complex, and 4i-complex systems were 18.25 ± 0.07 Å, 18.12 ± 0.07 Å, and 18.16 ± 0.08 Å, respectively ( Figure 5C). According to the observed behavior, the 4f-complex has a highly stiff structure against caspase-3. The compactness of the protein hydrophobic core was examined by calculating the protein's Solvent Accessible Surface Area (SASA). This was performed by measuring the surface area of the protein visible to the solvent, which is important for biomolecule stability [38]. The average SASA values for apo-protein, 4f-complex, and 4i-complex systems were 11,246 Å, 11,068 Å, and 11,174 Å, respectively ( Figure 5D). The SASA finding, when paired with the observations from the RMSD,

Molecular Dynamic and System Stability
A molecular dynamic simulation was carried out to predict the performance of the extracted compounds upon binding to the active site of protein as well as its interaction and stability through simulation [33,34]. The validation of system stability is essential to trace disrupted motions and avoid artifacts that may develop during the simulation. This study assessed Root-Mean-Square Deviation (RMSD) to measure system stability during the 20 ns simulations. The recorded average RMSD values for all frames of systems-apo-protein, 4f-complex, and 4i-complex systems-were 2.92 ± 0.56 Å, 2.04 ± 0.41 Å, and 2.49 ± 0.34 Å, respectively ( Figure 5A). These results revealed that the 4f-bound-to-protein complex system acquired a relatively more stable conformation than the other studied systems. During MD simulation, assessing protein structural flexibility upon ligand binding is critical for examining residue behavior and its connection with the ligand [35]. Protein residue fluctuations were evaluated using the Root-Mean-Square Fluctuation (RMSF) algorithm to evaluate the effect of inhibitor binding toward the respective targets over 20 ns simulations. The computed average RMSF values were 6.24 Å, 0.99 Å, and 4.70 Å for apo-protein, 4fcomplex, and 4i-complex systems, respectively. Overall residue fluctuations of individual systems are represented in Figure 5B. These values revealed that the 4f-bound-to-protein complex system has a lower residue fluctuation than the other systems. ROG was determined to evaluate overall system compactness as well as stability upon ligand binding during MD simulation [36,37]. The average Rg values for apo-protein, 4f-complex, and 4i-complex systems were 18.25 ± 0.07 Å, 18.12 ± 0.07 Å, and 18.16 ± 0.08 Å, respectively ( Figure 5C). According to the observed behavior, the 4f-complex has a highly stiff structure against caspase-3. The compactness of the protein hydrophobic core was examined by calculating the protein's Solvent Accessible Surface Area (SASA). This was performed by measuring the surface area of the protein visible to the solvent, which is important for biomolecule stability [38]. The average SASA values for apo-protein, 4f-complex, and 4icomplex systems were 11,246 Å, 11,068 Å, and 11,174 Å, respectively ( Figure 5D). The SASA finding, when paired with the observations from the RMSD, RMSF, and ROG computations, confirmed that the 4f-complex system remains intact inside the S2 domain binding site of caspase-3 receptors.
RMSF, and ROG computations, confirmed that the 4f-complex system remains intact inside the S2 domain binding site of caspase-3 receptors.

Binding Interaction Mechanism Based on Binding Free Energy Calculation
A popular method for determining the binding free energies of small molecules to biological macromolecules is the molecular mechanics' energy technique (MM/GBSA), which combines the generalized Born and surface area continuum solvation, and it may be more trustworthy than docking scores [39]. The MM-GBSA program in AMBER18 was used to calculate the binding free energies by extracting snapshots from the trajectories of the systems. As shown in Table 3

Binding Interaction Mechanism Based on Binding Free Energy Calculation
A popular method for determining the binding free energies of small molecules to biological macromolecules is the molecular mechanics' energy technique (MM/GBSA), which combines the generalized Born and surface area continuum solvation, and it may be more trustworthy than docking scores [39]. The MM-GBSA program in AMBER18 was used to calculate the binding free energies by extracting snapshots from the trajectories of the systems. As shown in Table 3, all the reported calculated energy components (except ∆G solv ) gave high negative values, indicating favorable interactions. The results indicate that the binding affinities of the 4f-complex and 4i-complex systems were −25.56 kcal/mol and −15.63 kcal/mol, respectively.
The interactions between the 4f and 4i compounds and the caspase-3 receptor protein residues are driven by the more positive electrostatic energy component, as shown by a detailed examination of each energy contribution, leading to the reported binding free energies. Substantial binding free energy values were observed in the gas phase for all the inhibition processes, with values up to −88.57 and −93.65 kcal/mol, respectively (Table 3).

Identification of the Critical Residues Responsible for Ligand Binding
The total energy involved when 4f and 4i compounds bind with these enzymes was further decomposed into the involvement of individual site residues to gain more knowledge about important residues involved in the inhibition of the S2 domain binding site receptor of caspase-3 receptors. From Figure 6  The interactions between the 4f and 4i compounds and the caspase-3 receptor protein residues are driven by the more positive electrostatic energy component, as shown by a detailed examination of each energy contribution, leading to the reported binding free energies. Substantial binding free energy values were observed in the gas phase for all the inhibition processes, with values up to −88.57 and −93.65 kcal/mol, respectively (Table 3).

Identification of the Critical Residues Responsible for Ligand Binding
The total energy involved when 4f and 4i compounds bind with these enzymes was further decomposed into the involvement of individual site residues to gain more knowledge about important residues involved in the inhibition of the S2 domain binding site receptor of caspase-3 receptors. From Figure 6

Ligand-Residue Interaction Network Profiles
One of the purposes of drug design is to make structural changes to therapeutic molecules to increase bioavailability, reduce toxicity, and improve pharmacokinetics [40].
The binding of receptor-specific active site residues to particular groups in the drug molecule results in the suppression of caspase-3, a key mediator of apoptotic cell death in mammals that cleaves over 500 cellular substrates to carry out the apoptosis program [41,42]. In light of the tight association between apoptosis and a wide range of disorders, caspase-3 inhibitors have the potential to pave the way for new treatments for immunodeficiency, Alzheimer's, Parkinson's, Huntington's, ischaemia, brain trauma, and amyotrophic lateral sclerosis [43]. It has been observed that the structural interactions of both compounds are hydrophobic and electrostatic in nature in the S2 domain binding site of the caspase-3 receptor. Figure 7 shows that the NH group of compound 4f's indazole ring occupied the S2 binding pocket via a secure network of H-bonds with Gly 94 and Glu95. Furthermore, Api-pi stacking was discovered between the Tyr 165 and Phe 217 residues and the pyrimidine ring. Additionally, the hot spot Arg 186 residue produced both π-cation and π-alkyl interactions with the phenyl and morpholine rings. Ultimately, a π-cation interaction (electrostatic interaction) between Met 33 and the phenyl ring of indazole was identified ( Figure 7A). Compound 4i, on the other hand, has developed a two π-cation contact with the indazole ring. Moreover, the trimethoxy ring has formed π-π stacking with Tyr 165. Eventually, Ala 134 formed a π-alkyl interaction with the pyrazole ring of indazole ( Figure  7B).

Ligand-Residue Interaction Network Profiles
One of the purposes of drug design is to make structural changes to therapeutic molecules to increase bioavailability, reduce toxicity, and improve pharmacokinetics [40].
The binding of receptor-specific active site residues to particular groups in the drug molecule results in the suppression of caspase-3, a key mediator of apoptotic cell death in mammals that cleaves over 500 cellular substrates to carry out the apoptosis program [41,42]. In light of the tight association between apoptosis and a wide range of disorders, caspase-3 inhibitors have the potential to pave the way for new treatments for immunodeficiency, Alzheimer's, Parkinson's, Huntington's, ischaemia, brain trauma, and amyotrophic lateral sclerosis [43]. It has been observed that the structural interactions of both compounds are hydrophobic and electrostatic in nature in the S2 domain binding site of the caspase-3 receptor. Figure 7 shows that the NH group of compound 4f's indazole ring occupied the S2 binding pocket via a secure network of H-bonds with Gly 94 and Glu95. Furthermore, Apipi stacking was discovered between the Tyr 165 and Phe 217 residues and the pyrimidine ring. Additionally, the hot spot Arg 186 residue produced both π-cation and π-alkyl interactions with the phenyl and morpholine rings. Ultimately, a π-cation interaction (electrostatic interaction) between Met 33 and the phenyl ring of indazole was identified ( Figure 7A). Compound 4i, on the other hand, has developed a two π-cation contact with the indazole ring. Moreover, the trimethoxy ring has formed π-π stacking with Tyr 165. Eventually, Ala 134 formed a π-alkyl interaction with the pyrazole ring of indazole ( Figure 7B).

In Silico ADMET Properties Prediction
A compound must meet the following requirements to be considered a prospective physiologically active molecule: (1) molecular weight < 500, (2) log P (lipophilicity) < 5, Based on the above criteria, the synthesized compounds were subjected to in silico tests for ADMET prediction for testing bioavailability and toxicity.

In Silico ADMET Properties Prediction
A compound must meet the following requirements to be considered a prospective physiologically active molecule: (1) molecular weight <500, (2)  Based on the above criteria, the synthesized compounds were subjected to in silico tests for ADMET prediction for testing bioavailability and toxicity. Table 4 shows the derived parameters for Lipinski's rule of five, topological polar surface area, aqueous solubility, and the number of rotatable bonds. The values for human intestinal absorption ranged from 88.003231 to 92.405990%, showing that the synthesized compounds had a moderate to good absorption capacity and supported their interaction with the target cell (Table 5).   Table 4 shows the derived parameters for Lipinski's rule of five, topological polar surface area, aqueous solubility, and the number of rotatable bonds. The values for human intestinal absorption ranged from 88.003231 to 92.405990%, showing that the synthesized compounds had a moderate to good absorption capacity and supported their interaction with the target cell (Table 5).   The in vitro Caco-2 cell permeability in the range of 0.727024-48.4113 nm/s and the in vitro MDCK cell permeability in the range of 0.268974-40.1723 nm/s characterized the synthesized compounds as having high permeability. The synthesized compounds have values ranging from 83.42 to 100.00%, indicating that they have a high affinity for proteins. The in vivo blood-brain barrier penetration ranges from 0.055 to 0.86, indicating that they have a low to moderate distribution in vivo, with medium to strong penetration capacity (Table 5). Bioactivity and toxicity risk values of synthesized compounds are shown in Table 6.

Structure-Activity Relationship
Concerning the data adopted in Tables 1 and 2, the presence of sulfadiazine of compound 4a or the trimethoxy groups of compound 4i at position 4 of the aniline ring resulted in its strong inhibitory activity and produced broad and potent antiproliferative activity against all of the three tested cell lines. However, the replacement of the hydrogen atom at position 5 of the pyrimidine ring in compound 4i with fluorine in compound 4c led to diminished activity. Meanwhile, the morpholino substituent at the para position of the aniline ring in compound 4f led to significant and selective cytotoxic activity against the MCF-7 cell line. The sulfonamide substituent in compounds 4d and 4g caused strong cytotoxic activity against MCF-7 cells that was twofold as potent as the standard, while strong activity was observed for these two molecules against Caco2 cell lines by about double or equal to the standard, respectively. Compound 4e, with the fluorine atom in position 5 of the pyrimidine ring along with the sulfanilamide group, illustrated potent antiproliferative activity toward the Caco2 cell line only. The sulfathiazole substituent in compounds 4h and 4b led to a lack of cytotoxic activity in all the three tested cell lines.

Conclusions
The authors synthesized and identified nine new indazol-pyrimidine derivatives according to different analyses. All the new compounds were evaluated for anticancer inhibitory activity against MCF-7, A549, and Caco2 human cancer cell lines. Five compounds possessed significant cytotoxic potential against MCF-7 cells and were more potent than the reference drug. From this, compounds 4f and 4i exhibited the lowest IC 50 values of 1.629 and 1.841 µM, respectively, compared with the reference drug with an IC 50 value of 8.029 µM. In addition, five products showed cytotoxic activity stronger than the standard against Caco2 cells. Moreover, two compounds evidenced potent antiproliferative activity that was stronger than the reference against the A549 cell line. Additionally, the most active products, 4f and 4i, were further examined for their mechanism of action by flow cytometry assay. It turns out that they were capable of activating caspase-3/7 and, therefore, inducing apoptosis. On the other hand, these two compounds demonstrated marked safety profiles toward human normal cells (MCF-10a), more than the reference, indicating that these compounds are more selective to cancerous cells relative to the reference. Consequently, the two promising candidates will be subjected to extensive future studies for in vivo animal models evaluation, and they can act as new compounds in developing new potent and highly safe anticancer products. We hope to produce highly effective, low-toxicity anticancer agents after the mandatory biological studies have been performed. Following that, the interaction's stability was assessed using a typical atomistic 20 ns dynamic simulation study. A number of parameters derived from MD simulation trajectories were computed and validated for the protein-ligand complex's stability under dynamic conditions. Prediction of computational drug-like properties showed that most of the synthesized compounds are safe with acceptable ADMET and druggable properties.

Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without further purification. When necessary, solvents were dried by standard methods. Melting points ( • C) were measured in open-glass capillaries using Branstead 9100 electrothermal melting point apparatus and are uncorrected. NMR spectra were obtained on a Bruker AC 500 Ultra Shield NMR spectrometer (Fällanden, Switzerland) at 500. 13 (Compounds 3a and b) A mixture of 2,4 dichloropyrimidine or 5-flouro-2,4 dichloropyrimidine (0.027 mol) and 5-aminoindazole (3.59 g, 0.027 mol) was dissolved in (8 mL) Ethanol with continuous stirring. Triethylamine (2.7 g, 0.027 mol) was added gradually, followed by refluxing the mixture at 80 • C for 4-6 h. After completion of the reaction (which was monitored by TLC), the formed precipitate was filtered off, washed with cold water, dried, and recrystallized from ethanol to afford 5-substituted-N-(2-chloropyrimidin-4-yl)-1H-indazol-5-amine according to the reported method [26].

General Procedure for Preparation of Compounds 4a-i
To a mixture of compound 3 (0.0018 mol) in butanol (25 mL), the appropriate aniline derivative (0.0018 mol) was added, followed by the addition of 4 drops of conc. HCl. The mixture was refluxed overnight, and after cooling, the formed precipitate was filtered off, washed with hot ethanol and/or ethyl acetate, and filtered off while hot, then recrystallized from ethanol to afford the desired compounds 4a-i.  The crystal structure of human caspase-3 was assessed at a resolution of 2.80 Å, which was retrieved from the protein data bank with codes 1GFW [44] and prepared using UCSF Chimera [45]. Using PROPKA, pH was fixed and optimized to 7.5 [46]. The extracted 2D structure was drawn using ChemBioDraw Ultra 12.1 [47]. The steepest descent approach and MMFF94 force field in Avogadro software [48] were used to optimize the 2D structure for energy minimization. In preparation for docking, hydrogen atoms were removed using UCSF chimera [45].

Molecular Docking
AutoDock Vina was used for docking calculations [49], and Gasteiger partial charges [50] were allocated during docking. The AutoDock graphical user interface offered by MGL tools was used to outline the AutoDock atom types [51]. The grid box was determined with grid parameters x = −36.6310, y = 37.0493, and z = 31.466 for the dimension and x = 15.9631, y = 14.398, and z = 10 for the central grid and exhaustiveness = 8. The Lamarckian genetic algorithm [52] was used to create docked conformations in descending order based on their docking energy.

Molecular Dynamic (MD) Simulations
The integration of Molecular Dynamic (MD) simulations in biological system studies enables exploring the physical motion of atoms and molecules that cannot be easily accessed by any other means [53]. The insight extracted from performing this simulation provides an intricate perspective into the biological systems' dynamic evolution, such as conformational changes and molecule association [53]. The MD simulations of all systems were performed using the GPU version of the PMEMD engine present in the AMBER 18 package [54]. The partial atomic charge of each compound was calculated with ANTECHAMBER's General Amber Force Field (GAFF) technique [55]. The Leap module of the AMBER 18 package implicitly solvated each system within an orthorhombic box of TIP3P water molecules within 10 Å of any box edge. The leap module was used to neutralize each system by incorporating Na + and Cl − counterions. A 2000-step initial minimization of each system was carried out in the presence of a 500 kcal/mol applied restraint potential, followed by a 1000-step full minimization using the conjugate gradient algorithm without restraints. During the MD simulation, each system was gradually heated from 0 K to 300 K over 500 ps, ensuring that all systems had the same amount of atoms and volume. The system's solutes were subjected to a 10 kcal/mol potential harmonic constraint and a 1 ps collision frequency. Following that, each system was heated and equilibrated for 500 ps at a constant temperature of 300 K. To simulate an isobaric-isothermal (NPT) ensemble, the number of atoms and pressure within each system for each production simulation were kept constant, with the system's pressure maintained at 1 bar using the Berendsen barostat [56]. For 20 ns, each system was MD simulated. The SHAKE method was used to constrain the hydrogen bond atoms in each simulation. Each simulation used a 2fs step size and integrated an SPFP precision model. An isobaric-isothermal ensemble (NPT) with randomized seeding, constant pressure of 1 bar, pressure-coupling constant of 2 ps, temperature of 300 K, and a Langevin thermostat with a collision frequency of 1 ps was used in the simulations.

Post-MD Analysis
After saving the trajectories obtained by MD simulations every 1 ps, the trajectories were analyzed using the AMBER18 suite's CPPTRAJ [57] module. The Origin [58] data analysis program and Chimera [45] were used to create all graphs and visualizations.

Thermodynamic Calculation
The Poisson-Boltzmann or generalized Born and surface area continuum solvation (MM/PBSA and MM/GBSA) approach is useful in the estimation of ligand-binding affinities [59][60][61]. The Protein-Ligand complex molecular simulations used by MM/GBSA and MM/PBSA compute rigorous statistical-mechanical binding free energy within a defined force field. Binding free energy averaged over 200 snapshots extracted from the entire 20 ns trajectory. The estimation of the change in binding free energy (∆G) for each molecular species (complex, ligand, and receptor) can be represented as follows: [62].
G sol = G GB + G SA (4) The terms E gas , E int , E ele , and E vdw symbolize the gas-phase energy, internal energy, Coulomb energy, and van der Waals energy. The E gas was directly assessed from the FF14SB force field terms. Solvation free energy (G sol ) was evaluated from the energy involvement from the polar states (G GB ) and non-polar states (G). The non-polar solvation free energy (G SA ) was determined from the Solvent Accessible Surface Area (SASA) [63,64] using a water probe radius of 1.4 Å. In contrast, solving the GB equation assessed the polar solvation (G GB ) contribution. Items S and T symbolize the total entropy of the solute and temperature, respectively. The MM/GBSA-binding free energy method in Amber18 was used to calculate the contribution of each residue to the total binding free energy.

Computation of Drug-like Parameters and ADMET Profiling
The online tool kit Molinspiration (http://www.molinspiration.com/ (accessed on 16 April 2023)) and the OSIRIS property explorer were used to compute drug-like features from the above-mentioned compounds' 2D chemical structures [65][66][67].